Project Summary Actin is a key protein for normal cell function, and the ability to control the polymerization of actin filaments is essential in the cell. Actin assembles into filaments with two distinct ends, and most of this control happens at what is termed the plus or barbed end of the filament. Two key proteins function at the barbed end: capping protein and formins. As suggested by its name, capping protein ?caps? the barbed end and prevents further polymerization, but the activity of capping protein is also regulated by the action of proteins such as myotrophin/V-1, CARMIL and twinfilin. Formins have the opposite effect of capping protein and enhance polymerization at the barbed end. However, just as capping protein is regulated, the function of the formin INF2 is modulated by cyclase-associated protein (CAP) as well as post-translational modifications to actin. The focus of this proposal is to understand the structural, dynamical and functional mechanisms that regulate the barbed end of the filament. Through a combination of computational, in vitro and in vivo studies we will: (a) gain new understanding of what defines the barbed end and how it is affected by the nucleotide state of actin; (b) learn how capping protein interacts with the barbed end and how this interaction is affected by the steric and allosteric effects of V-1, CARMIL and twinfilin; and (c) determine how INF2 interacts with the barbed and how both CAP and lysine methylation of actin alter this interaction.